Ischemic Heart Disease/Myocardial Infarction
Ischemic heart disease is the most common cause of death in the world. In the United States alone an estimated 785,000 people will have a myocardial infarction (MI) each year; approximately 1 per minute [Roger V L, Go A S, Lloyd-Jones D M, Benjamin E J, Berry J D, Borden W B, et al. Executive summary: heart disease and stroke statistics—2012 update: a report from the American Heart Association. Circulation. 2012; 125:188-97]. The adverse remodeling that occurs after myocardial infarction contributes to the impaired cardiac function and heart failure associated with increased morbidity and mortality. Advances made in interventional, largely early reperfusion therapies, have improved patient survival while increasing the morbidity and mortality of the resulting heart failure [Pfeffer M A, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81:1161-72; Opie L H, Commerford P J, Gersh B J, Pfeffer M A. Controversies in ventricular remodeling. Lancet. 2006; 367:356-67; Dorn G W, 2nd. Novel pharmacotherapies to abrogate post-infarction ventricular remodeling. Nat Rev Cardiol. 2009; 6:283-91]. The size of the infarcted area, the infarcted wound healing, and chronic left ventricular (LV) remodeling determine the extent of heart failure that results [Pfeffer M A, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81:1161-72; Opie L H, Commerford P J, Gersh B J, Pfeffer M A. Controversies in ventricular remodelling. Lancet. 2006; 367:356-67; Dorn G W, 2nd. Novel pharmacotherapies to abrogate post-infarction ventricular remodeling. Nat Rev Cardiol. 2009; 6:283-91].
Ischemia
The myocardium depends almost entirely on aerobic metabolism, since oxygen stores in the heart are meager. Myocardial oxygen supply rises and falls in response to the oxygen (energy) demands of the myocardium. The term “autoregulation” refers to the ability to maintain myocardial perfusion at constant levels in the face of changing driving forces. Autoregulation maintains coronary perfusion at relatively constant levels over a wide range of mean aortic pressure. When aortic pressure exceeds its upper or lower limits, coronary blood flow precipitously declines or increases proportionately.
The heart needs to be supplied with a sufficient quantity of oxygen to prevent under-perfusion. When reduced perfusion pressure distal to stenoses is not compensated by auto-regulatory dilation of the resistance vessels, ischemia, meaning a lack of blood supply and oxygen, occurs. Because the zone least supplied generally is the farthest out, ischemia generally appears in areas farthest away from the blood supply.
After total or near-total occlusion of a coronary artery, myocardial perfusion occurs by way of collaterals, meaning vascular channels that interconnect epicardial arteries. Collateral channels may form acutely or may preexist in an under-developed state before the appearance of coronary artery disease. Preexisting collaterals are thin-walled structures ranging in diameter from 20 μm to 200 μm, with a variable density among different species. Preexisting collaterals normally are closed and nonfunctional, because no pressure gradient exists to drive flow between the arteries they connect. After coronary occlusion, the distal pressure drops precipitously and preexisting collaterals open virtually instantly.
The term “myocardial ischemia” refers to a decrease in blood supply and oxygen to the cells of the myocardium. The development of myocardial ischemia has been attributed to two mechanisms: (1) increased myocardial oxygen demand, and (2) decreased myocardial perfusion and oxygen delivery [Willerson, J. T. et al., J. Am. Coll. Cardiol. 8(1): 245-50 (1986)]. Myocardial ischemia generally appears first and is more extensive in the subendocardial region, since these deeper myocardial layers are farthest from the blood supply, with greater need for oxygen.
Transient Ischemia
The term “transient ischemia” as used herein refers to a reversible (meaning that the myocytes survive the insult) narrowing of a coronary artery at rest or with exercise where there is no thrombus or plaque rupture but where blood supply cannot be met. Every time the heart's oxygen demand increases, an imbalance between oxygen demand and supply is created. Transient ischemia produces a cascade of events beginning with metabolic and biochemical alterations leading to impaired ventricular relaxation and diastolic dysfunction, impaired systolic function, and electrocardiographic abnormalities with ST segment alterations, followed by increased end-diastolic pressure with left ventricular dyssynchrony, hypokineses, akinesis, and dyskinesis, and lastly painful symptoms of angina. Even though ischemic myocytes experience physiological and metabolic changes within seconds of the cessation of coronary flow, resulting in T wave and sometimes ST segment abnormalities (but without serum enzyme elevation), no cell death results from the ischemia [Kloner, R. A. and Jennings, R B, Circulation 104: 2981-89 (2001)]. Once blood flow is re-established, a complete recovery of myocyte contractile function takes place.
Although angina pectoris (chest pain) may be a symptom of transient ischemia, by and large transient ischemia is silent (meaning ST-segment depression of at least 1 mm is present without associated symptoms, e.g., chest pain) in 79% of subjects. In most patients with stable angina, for example, physical effort or emotion, with a resultant increase in heart rate, blood pressure, or contractile state, or any combination thereof, increases myocardial oxygen demand without an adequate delivery in oxygen delivery through tightly narrowed (stenosed) coronary arteries. More than 40% of patients with stable angina treated with one or more antianginal drugs have frequent episodes of silent ischemia, which has been shown to predict a higher risk of coronary events and cardiac death [Deedwania, P C, Carbajal, E V, Arch. Intern. Med. 150: 2373-2382 (1991)].
Chronic Myocardial Ischemia
The term “chronic myocardial ischemia (CMI)” as used herein refers to a prolonged subacute or chronic state of myocardial ischemia due to narrowing of a coronary blood vessel in which the myocardium “hibernates”, meaning that the myocardium downregulates or reduces its contractility, and hence its myocardial oxygen demand, to match reduced perfusion, thereby preserving cellular viability and preventing myocardial necrosis. This hibernating myocardium is capable of returning to normal or near-normal function on restoration of an adequate blood supply. Once coronary blood flow has been restored to normal or near normal and ischemia is resolved, however, the hibernating myocardium still does not contract. This flow-function mismatch resulting in a slow return of cardiac function after resolution of ischemia has been called stunning. The length of time for function to return is quite variable, ranging from days to months, and is dependent on a number of parameters, including the duration of the original ischemic insult, the severity of ischemia during the original insult, and the adequacy of the return of the arterial flow. A number of studies have provided evidence for inflammation in hibernating myocardium [Heusch, G. et al., Am. J. Physiol. Heart Circ. Physiol. 288: 984-99 (2005)]. A study conducted in a porcine model of myocardial hibernation in which the mean rest (left anterior descending coronary artery (LAD) coronary blood flow was reduced to about 60% of baseline for a period of 24 hours to four weeks, detected apoptotic myocytes in all experimental pigs in the hibernating regions supplied by the stenotic LAD, suggesting that functional downregulation may not be adequate to prevent gradual, ongoing myocyte death through apoptosis in hibernating myocardium [Chen, C, et al., J. Am. Coll. Cardiol. 30: 1407-12 (1997)].
Acute Myocardial Infarction (AMI)
Another type of insult occurs during AMI. AMI is an abrupt change in the lumen of a coronary blood vessel that results in ischemic infarction, meaning that it continues until heart muscle dies. On gross inspection, myocardial infarction can be divided into two major types: transmural infarcts, in which the myocardial necrosis involves the full or nearly full thickness of the ventricular wall, and subendocardial (non-transmural) infarcts, in which the myocardial necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium. There often is total occlusion of the vessel with ST segment elevation because of thrombus formation within the lumen as a result of plaque rupture. The prolonged ischemic insult results in apoptotic and necrotic cardiomyocyte cell death [See Kajstura, J., et al., Lab Invest. 74: 86-107 (1996)]. Necrosis compromises the integrity of the sarcolemmal membrane and intracellular macromolecules such that serum cardiac markers, such as cardiac-specific troponins and enzymes, such as serum creatine kinase (CK), are released. In addition, the patient may have electrocardiogram (ECG) changes because of full thickness damage to the muscle. An ST-Elevation Myocardial Infarction (STEMI) is a larger injury than a non-ST-elevation myocardial infarction. ST-segment elevation and Q waves on the ECG, two features highly indicative of myocardial infarction, are seen in only about half of myocardial infarction cases on presentation.
AMI remains common with a reported annual incidence of 1.1 million cases in the United States alone [Antman, E. M., Braunwald, E., Acute Myocardial Infarction, in Principles of Internal Medicine, 15th Ed., Braunwald, E. et al., Eds., New York: McGraw-Hill (2001)]. Preclinical and clinical data demonstrate that following a myocardial infarction, the acute loss of myocardial muscle cells and the accompanying peri-infarct border zone hypo-perfusion result in a cascade of events causing an immediate diminution of cardiac function, with the potential for long term persistence. The extent of myocardial cell loss is dependent on the duration of coronary artery occlusion, existing collateral coronary circulation and the condition of the cardiac microvasculature [Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheilban, I. e. al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M. R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13 (1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002); Hirai, T. et al., Circulation 79: 791-96 (1989); Ejiri, M. et al., J. Cardiology 20: 31-37 (1990)]. Because myocardial cells have virtually no ability to regenerate, myocardial infarction leads to permanent cardiac dysfunction due to contractile-muscle cell loss and replacement with nonfunctioning fibrotic scarring [Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002)]. Moreover, compensatory hypertrophy of viable cardiac muscle leads to microvascular insufficiency, which results in further demise in cardiac function by causing myocardial muscle hibernation and apoptosis of hypertrophied myocytes in the peri-infarct border zone.
Among survivors of myocardial infarction, residual cardiac function is influenced by the extent of ventricular remodeling (meaning changes in size, shape, and function, typically a progressive decline in function, of the heart after injury). Alterations in ventricular topography (meaning the shape, configuration, or morphology of a ventricle) occur in both infarcted and healthy cardiac tissue after myocardial infarction [Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990)]. Ventricular dilatation (meaning a stretching, enlarging or spreading out of the ventricle) causes a decrease in global cardiac function and is affected by the infarct size, infarct healing and ventricular wall stresses. Recent efforts to minimize remodeling have been successful by limiting infarct size through rapid reperfusion (meaning restoration of blood flow) using thromobolytic agents, and mechanical interventions, including, but not limited to, placement of a stent, along with reducing ventricular wall stresses by judicious use of pre-load therapies and proper after-load management [Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990)]. Regardless of these interventions, a substantial percentage of patients experience clinically relevant and long-term cardiac dysfunction after myocardial infarction [Sheiban, I. et al., J. Am. Coll. Cardiol. 38: 464-71 (2001)]. Despite revascularization of the infarct related artery circulation and appropriate medical management to minimize ventricular wall stresses, a significant percentage of these patients experience ventricular remodeling, permanent cardiac dysfunction, and consequently remain at an increased lifetime risk of experiencing adverse cardiac events, including death [Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990)].
At the cellular level, immediately following a myocardial infarction, transient generalized cardiac dysfunction uniformly occurs. In the setting of a brief (i.e., lasting three minutes to five minutes) coronary artery occlusion, energy metabolism is impaired, leading to demonstrable cardiac muscle dysfunction that can persist for up to 48 hours despite immediate reperfusion. This so-called “stunned myocardium phenomenon” occurs subsequent to or after reperfusion and is thought to be a result of reactive oxygen species. The process is transient and is not associated with an inflammatory response [Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002)]. After successful revascularization, significant recovery from stunning occurs within three to four days, although complete recovery may take much longer [Boli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K. et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al., Lancet 364: 141-48 (2004)].
Inflammation
Coronary artery occlusion of more significant duration, i.e., lasting more than five minutes, leads to myocardial ischemia (i.e. an insufficient blood flow to the heart's muscle mass) and is associated with a significant inflammatory response that begins immediately after reperfusion and can last for up to several weeks [Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. et al., Circulation 98: 687-798 (1998)]. The inflammatory process following reperfusion is complex. Initially it contributes to myocardial damage but later leads to healing and scar formation. This complex process appears to occur in two phases. In the first so-called “hot” phase (within the first five days), reactive oxygen species (in the ischemic myocardial tissue) and complement activation generate a signal chemotactic for leukocytes (chemotaxis is the directed motion of a motile cell, organism or part towards environmental conditions it deems attractive and/or away from surroundings it finds repellent) and initiate a cytokine cascade [Lefer, D. J., Granger, D. N., Am. J. Med. 4:315-23 (2000); Frangogiannis, N. G., et al., Circulation 7:699-710 (1998)]. Mast cell degranulation, tumor necrosis factor alpha (TNFα) release, and increased interleukin-6 (IL-6), intercellular adhesion molecule 1 (“ICAM-1” or CD-54, a receptor typically expressed on endothelial cells and cells of the immune system), selectin (L, E and P) and integrin (CD11a, CD11b and CD18) expression all appear to contribute to neutrophil accumulation and degranulation in ischemic myocardium [Frangogiannis, N. G. et al., Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc. Natl Acad. Sci USA. 10: 5456-61 (2000); Lasky, L. A., Science 258: 964-69 (1992); Ma, X. L., et al., Circulation 88(2): 649-58 (1993); Simpson, P. J. et al., J. Clin. Invest. 2: 624-29 (1998)]. Neutrophils contribute significantly to myocardial cell damage and death through microvascular obstruction and activation of neutrophil respiratory burst pathways after ligand-specific adhesion to cardiac myocytes [Entman, M. L., et al., J. Clin. Invest. 4: 1335-45 (1992)]. During the “hot” phase, angiogenesis is inhibited due to the release of angiostatic substances, including interferon gamma-inducible protein (IP 10) [Frangogiannis, N. G., et al., FASEB J. 15: 1428-30 (2001)].
In the second phase, the cardiac repair process begins (about day 6 to about day 14), which eventually leads to scar formation (about day 14 to about day 21) and subsequent ventricular remodeling (about day 21 to about day 90). Soon after reperfusion, monocytes infiltrate the infarcted myocardium. Attracted by complement (C5a), transforming growth factor B1 (“TGF-β1”) and monocyte chemotactic protein 1 (“MCP-1”), monocytes differentiate into macrophages that initiate the healing process by scavenging dead tissue, regulating extracellular matrix metabolism, and inducing fibroblast proliferation [Birdshall, H. H., et al., Circulation 3: 684-92 (1997)]. Secretion of interleukin 10 (IL-10) by infiltrating lymphocytes also promotes healing by down-regulating inflammatory cytokines and influencing tissue remodeling [Frangogiannis, N. G. et al., J. Immunol. 5:2798-2808 (2000)]. Mast cells also appear to be involved in the later stages of myocardial repair by participating in the formation of fibrotic scar tissue. Stem Cell Factor (SCF) is a potent attractor of mast cells. SCF mRNA has been shown to be up-regulated in ischemic myocardial segments in a canine model of myocardial infarction and thus may contribute to mast cell accumulation at ischemic myocardial sites [Franigogiannis, N. G. et al., Circulation 98: 687-798 (1998)]. Mast cell products (including TGF-β, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and gelatinases A and B) induce fibroblast proliferation, influence extracellular matrix metabolism, and induce angiogenesis [Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., et al., Cardiology 93: 168-74 (2000)].
Initiation of the Inflammatory Process
Complement Activation
Hill and Ward [Hill J H, Ward P A. The phlogistic role of C3 leukotactic fragment in myocardial infarcts of rats. J Exp Med 1971; 885-890] were the first to demonstrate that ischemic myocardial injury can activate the complement cascade in a rat model of myocardial infarction. Subsequently, Pinckard et al. [Pinckard R. N., Olson M. S., Giclas P. C., et al. Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarction. J Clin Invest 1975; 3:740-750] suggested that myocardial cell necrosis results in the release of subcellular membrane constituents rich in mitochondria, which are capable of triggering the early acting components (C1, C4, C2 and C3) of the complement cascade. Rossen et al. [Rossen R. D., Michael L. H., Hawkins H. K., et al. Cardiolipin-protein complexes and initiation of complement activation after coronary artery occlusion. Circ Res 1994; 3:546-555] have suggested that during myocardial ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments rich in cardiolipin and protein. By binding C1 and supplying sites for the assembly of later acting complement components, these subcellular fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury. mRNA and proteins for all the components of the classical complement pathway are up-regulated in areas of myocardial infarcts [Vakeva A. P., Agah A., Rollins S. A., et al. Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-05 therapy. Circulation 1998; 22:2259-2267; Yasojima K., Kilgore K. S., Washington R. A., Lucchesi B. R., McGeer P. L. Complement gene expression by rabbit heart: up-regulation by ischemia and reperfusion. Circ Res 1998; 11:1224-1230].
Complement activation may play an important role in mediating neutrophil and monocyte recruitment in the injured myocardium. Dreyer et al. [Dreyer W. J., Michael L. H., Nguyen T., et al. Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury. Circ Res 1992; 6:1518-1524] showed that post-ischemic cardiac lymph contains leukocyte chemotactic activity, which is maximal during the first hour of reperfusion with washout within the next 3 hours.
Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) are molecules with unpaired electrons in their outer orbit. They have the potential to directly injure cardiac myocytes and vascular cells and may be involved in triggering inflammatory cascades through the induction of cytokines [Lefer D. J., Granger D. N. Oxidative stress and cardiac disease. Am J Med 2000; 4:315-323; Dhalla N. S., Elmoselhi A. B., Hata T., Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res 2000; 3:446-456]. Reactive oxygen species have been shown to exert a direct inhibitory effect on myocardial function in vivo and have a critical role in the pathogenesis of myocardial stunning [Bolli R. Oxygen-derived free radicals and postischemic myocardial dysfunction (‘stunned myocardium’). J Am Coll Cardiol 1988; 1:239-249]. In addition, evidence exists for a potential role of reactive oxygen in leukocyte chemotaxis [Granger D. N. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1988; 6(2):H1269-H1275]. Potential mechanisms through which reactive oxygen intermediates may generate a leukotactic stimulus include complement activation, induction of P-selectin expression, chemokine upregulation, and an increase in the ability of endothelial ICAM-1 to bind to neutrophils [Shingu M., Nobunaga M. Chemotactic activity generated in human serum from the fifth component of complement by hydrogen peroxide. Am J Pathol 1984; 2:201-206; Akgur F. M., Brown M. F., Zibari G. B., et al. Role of superoxide in hemorrhagic shock-induced P-selectin expression. Am J Physiol Heart Circ Physiol 2000; 2:H791-H797; Patel K. D., Zimmerman G. A., Prescott S. M., McEver R. P., McIntyre T. M. Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils. J Cell Biol 1991; 4:749-759; Lakshminarayanan V., Drab-Weiss E. A., Roebuck K. A. H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappa B to the interleukin-8 promoter in endothelial and epithelial cells. J Biol Chem 1998; 49:32670-32678; Lakshminarayanan V., Beno D. W., Costa R. H., Roebuck K. A. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem 1997; 52:32910-32918; Sellak H., Franzini E., Hakim J., Pasquier C. Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable up-regulation. Blood 1994; 9:2669-2677].
Most of the evidence implicating ROS in the pathophysiology of myocardial infarction is derived from investigations using free radical scavengers. Jolly et al. [Jolly S. R., Kane W. J., Bailie M. B., Abrams G. D., Lucchesi B. R. Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984; 3:277-285] demonstrated that the combination of the antioxidant enzymes superoxide dismutase and catalase significantly reduced infarct size in dogs undergoing experimental myocardial ischemia and reperfusion. Other investigators found similar beneficial effects of antioxidant interventions in experimental models of myocardial infarction. However, there is a significant number of studies describing a failure of antioxidants to prevent injury or demonstrating an early protective effect, which waned with increased duration of reperfusion [Uraizee A., Reimer K. A., Murry C. E., Jennings R. B. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation 1987; 6:1237-1248; Gallagher K. P., Buda A. J., Pace D., Gerren R. A., Shlafer M. Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation 1986; 5:1065-1076; Richard V. J., Murry C. E., Jennings R. B., Reimer K. A. Therapy to reduce free radicals during early reperfusion does not limit the size of myocardial infarcts caused by 90 minutes of ischemia in dogs. Circulation 1988; 2:473-480]. Recently, transgenic mice that overexpress superoxide dismutase (SOD1) showed significant protection from post-ischemic injury and a significant decrease in infarct size in Langendorf-perfused hearts undergoing left coronary artery ligation [Wang P., Chen H., Qin H., et al. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents post-ischemic injury. Proc Natl Acad Sci USA 1998; 8:4556-4560; Chen Z., Siu B., Ho Y. S., et al. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol 1998; 11:2281-2289]. Unfortunately, two clinical studies using recombinant human superoxide dismutase in patients with acute myocardial infarction undergoing thrombolysis [Murohara Y., Yui Y., Hattori R., Kawai C. Effects of superoxide dismutase on reperfusion arrhythmias and left ventricular function in patients undergoing thrombolysis for anterior wall acute myocardial infarction. Am J Cardiol 1991; 8:765-767.] or balloon angioplasty [Flaherty J. T., Pitt B., Gruber J. W., et al. Recombinant human superoxide dismutase (h-SOD) fails to improve recovery of ventricular function in patients undergoing coronary angioplasty for acute myocardial infarction. Circulation 1994; 5:1982-1991] demonstrated no significant improvement in left ventricular function. In addition, prolonged coronary occlusion (>2 h) is usually present in the clinical setting of reperfused myocardial infarction and may cause extensive irreversible myocardial damage, leaving fewer myocytes to be affected by free radical-mediated injury [Lefer D. J., Granger D. N. Oxidative stress and cardiac disease. Am J Med 2000; 4:315-323; Maxwell S. R., Lip G. Y. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int J Cardiol 1997; 2:95-117].
Meldrum et al. [Meldrum D R, Dinarello C A, Cleveland J C, Jr, et al. Hydrogen peroxide induces tumor necrosis factor alpha mediated cardiac injury by a p38 mitogen activated protein kinase dependent mechanisms. Surgery. 1998; 124:291-296. discussion 297] demonstrated that H2O2 alone induced myocardial TNF-α mediated cardiac injury by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. It has been hypothesized that reactive oxygen intermediates may generate a leukotatic stimulus that includes, complement activation, induction of hemorrhagic shock-induced P-selectin expression, chemokine up-regulation and an increase in the endothelial intercellular adhesion molecule (ICAM)-1 ability to bind neutrophils [Shingu M, Nobunaga M. Chemotactic activity generated in human serum from the fifth component of complement by hydrogen peroxide. Am J Pathol. 1984; 117:201-206; Akgur F M, Brown M F, Zibari G B, et al. Role of superoxide in hemorrhagic shock-induced P-selectin expression. Am J Physiol Heart Circ Physiol. 2000; 279:H791-H797; Lakshminarayanan V, Beno D W, Costa R H, Roebuck K A. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem. 1997; 272:32910-32918; Sellak H, Franzini E, Hakim J, Pasquier C. Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable up-regulation. Blood. 1994; 83:2669-2677]. It was reported that the use of the antioxidant enzymes superoxide dismutase and catalase reduced infarct size in dogs with myocardial ischemia and reperfusion [Jolly S R, Kane W J, Bailie M B, Abrams G D, Lucchesi B R. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res. 1984; 54:277-285]. However, failed studies have been reported where antioxidant treatment was used to prevent myocardial ischemic injury [Uraizee A, Reimer K A, Murry C E, Jennings R B. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation. 1987; 75:1237-1248; Gallagher K P, Buda A J, Pace D, Gerren R A, Shlafer M. Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation. 1986; 73:1065-1076]. For example, two clinical studies in which recombinant human superoxide dismutase was used in patients with an acute myocardial infarction undergoing percutaneous coronary intervention or thrombolysis showed no significant improvement of left ventricular function [Murohara Y, Yui Y, Hattori R, Kawai C. Effects of superoxide dismutase on reperfusion arrhythmias and left ventricular function in patients undergoing thrombolysis for anterior wall acute myocardial infarction. Am J Cardiol. 1991; 67:765-767; Flaherty J T, Pitt B, Gruber J W, et al. Recombinant human superoxide dismutase (h-SOD) fails to improve recovery of ventricular function in patients undergoing coronary angioplasty for acute myocardial infarction. Circulation. 1994; 89:1982-1991].
Cytokine Cascade
Experimental myocardial infarction is associated with the coordinated activation of a series of cytokine and adhesion molecule genes. A critical element in the regulation of these genes involves the complex formed by NF-κB and Iκβ [Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 1989; 2: 227-229]. NF-κB is activated by a vast number of agents, including cytokines (such as TNF-α and IL-1β) and free radicals. Cytokines can self-amplify through a positive feedback loop targeting the nuclear factor (NF)-κB. Up-regulation of TNF-α in the infarct myocardium can up-regulate the levels of TNF-α in the neighboring normal myocardium, leading to amplified cytokine effects [Irwin M, Mak S, Mann D L, et al. Tissue expression and immunolocalization of tumor necrosis factor-alpha in post infarction-dysfunctional myocardium. Circulation. 1999; 99:1492-1498]. TNF-α stimulates expression of pro-inflammatory cytokines, chemokines and adhesion molecules by leukocytes and endothelial cells, and regulates extracellular matrix metabolism by reducing collagen synthesis and by enhancing matrix metalloprotease (MMP) activity in cardiac fibroblasts; other adhesive cytokines, such as monocyte chemoattractant protein (MCP)-1, are also induced in the ischemic and re-perfused canine myocardium [Siwik D A, Chang D L, Coluci W S. Interleukin-1 beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro. Circ Res. 2000; 86:1259-1265]. Kumar et al. [Kumar A G, Ballantyne C M, Michael L H, et al. Induction of monocyte chemoattractant protein-1 in the small veins of the ischemic and re-perfused canine myocardium. Circulation. 1997; 95:693-700] suggested that MCP-1 μlays a significant role in monocyte trafficking in re-perfused myocardium.
The mechanisms responsible for triggering the cytokine cascade in the infarcted myocardium have only recently been investigated. Several studies have indicated a role for preformed mast cell-derived mediators in initiating the cytokine cascade ultimately responsible for ICAM-1 induction in the re-perfused canine myocardium [Frangogiannis N. G., Lindsey M. L., Michael L. H., et al. Resident cardiac mast cells de-granulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 1998; 7:699-710; Frangogiannis N. G., Entman M. L. Mast cells in myocardial ischemia and reperfusion, Mast cells and basophils in physiology, pathology and host defense. In: Marone G., Liechtenstein L. M., Galli S. J., editors. London: Academic Press; 2000. p. 507-522; Frangogiannis N. G., Burns A. R., Michael L. H., Entman M. L. Histochemical and morphological characteristics of canine cardiac mast cells. Histochem J 1999; 4:221-229]. Mast cells have been recognized as an important source of preformed and newly synthesized cytokines, chemokines and growth factors [Frangogiannis N. G., Lindsey M. L., Michael L. H., et al. Resident cardiac mast cells de-granulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 1998; 7:699-710; Frangogiannis N. G., Entman M. L. Mast cells in myocardial ischaemia and reperfusion, Mast cells and basophils in physiology, pathology and host defense. In: Marone G., Liechtenstein L. M., Galli S. J., editors. London: Academic Press; 2000. p. 507-522; Frangogiannis N. G., Burns A. R., Michael L. H., Entman M. L. Histochemical and morphological characteristics of canine cardiac mast cells. Histochem J 1999; 4:221-229]. Gordon and Galli [Gordon J R, Galli S J. Mast cells as a source of both preformed and immunologically inducible TNF-alpha/cachectin. Nature 1990; 274-276; Gordon J. R., Burd P. R., Galli S. J. Mast cells as a source of multifunctional cytokines Immunol Today 1990; 12:458-464] identified mouse peritoneal mast cells as an important source of both preformed and immunologically-induced TNF-α. The constitutive presence of TNF-α in canine cardiac mast cells have led to the hypothesis that mast cell-derived TNF-α may be released following myocardial ischemia, representing an ‘upstream’ cytokine responsible for initiating the inflammatory cascade [Frangogiannis N G, Cardiovascular Research (2002) Vol. 53, Issue 1, pp. 31-47].
Moreover, it has been reported that early post-ischemic cardiac lymph is capable of inducing IL-6 expression in canine mononuclear cells in vitro. Incubation with a neutralizing antibody to TNF-α in part inhibited IL-6 up-regulation, suggesting an important role for TNF-α as the upstream cytokine inducer. Mast cell degranulation appears to be confined in the ischemic area and results in rapid release of TNF-α, inducing IL-6 in infiltrating mononuclear cells [Frangogiannis N G, Cardiovascular Research (2002) Vol. 53, Issue 1, pp. 31-47; 56, 61].
The role of TNF-α in myocardial infarction is thought to be more complex than simply serving as a trigger of a cytokine cascade [Sack M. N., Smith R. M., Opie L. H. Tumor necrosis factor in myocardial hypertrophy and ischemia—an anti-apoptotic perspective. Cardiovasc Res 2000; 3:688-695; Belosjorow S., Schulz R., Dorge H., Schade F. U., Heusch G. Endotoxin and ischemic preconditioning: TNF-alpha concentration and myocardial infarct development in rabbits. Am J Physiol 1999; 6(2):H2470-H2475]. Recent experiments using TNFR1/TNFR2 double receptor knockout mice undergoing left coronary artery ligation had significantly larger infarct size and increased myocyte apoptosis when compared with wild-type controls [Kurrelmeyer K. M., Michael L. H., Baumgarten G., et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci USA 2000; 10:5456-5461]. These findings suggested that TNF-α may induce a cytoprotective signal capable of preventing or delaying the development of myocyte apoptosis following myocardial infarction.
Other studies have shown that TNF-α expression during the healing phase was not confined to the infarct or peri-infarct zone, but was also localized in the normal non-infarcted myocardium, in which remodeling was ongoing. Thus, sustained TNF-α expression may have a role in the reparative process following myocardial infarction [Irwin M. W., Mak S., Mann D. L., et al. Tissue expression and immunolocalization of tumor necrosis factor-alpha in post-infarction dysfunctional myocardium. Circulation 1999; 11:1492-1498; Jacobs M., Staufenberger S., Gergs U., et al. Tumor necrosis factor-alpha at acute myocardial infarction in rats and effects on cardiac fibroblasts. J Mol Cell Cardiol 1999; 11:1949-1959].
Cytokine and Chemokine Upregulation
Chemokine up-regulation is a noted feature of the post-infarction inflammatory response (Table 1) [Frangogiannis N G. Chemokines in ischemia and reperfusion. Thromb Haemost. 2007; 97:738-747]. Investigators have demonstrated strong induction of several chemokines in the ischemic myocardium, supporting their role in leukocyte recruitment [Birdsall H H, Green D M, Trial J, et al. Complement C5a, TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion. Circulation. 1997; 95:684-692]. MCP-1 up-regulation has been demonstrated in a mouse model [Tarzami S T, Cheng R, Miao W, Kitsis R N, Berman J W. Chemokine expression in myocardial ischemia: MIP-2 dependent MCP-1 expression protects cardiomyocytes from cell death. J Mol Cell Cardiol. 2002; 34:209-221]. Frangogiannis reported that a MCP-1−/− infarct mouse model had decreased messenger ribonucleic acid (mRNA) expression of the cytokines TNF-α, IL-1β, TGF-β and IL-10, and showed defective macrophage differentiation [Frangogiannis N G. Chemokines in ischemia and reperfusion. Thromb Haemost. 2007; 97:738-747]. Cytokines, such as TNF-α and IL-6, are rapidly released in the central zone during a myocardial infarction; however, they are usually maximal in the border zone [Irwin M, Mak S, Mann D L, et al. Tissue expression and immunolocalization of tumor necrosis factor-alpha in post infarction-dysfunctional myocardium. Circulation. 1999; 99:1492-1498; Gwechenberger M, Mendoza L H, Youker K A, et al. Cardiac myocytes produce interleukin-6 in culture and in viable border zone of re-perfused infarctions. Circulation. 1999; 99:546-551]. This robust up-regulation may return to baseline levels if the infarction is small; if the infarction is large and the inflammatory response is excessive, there can be sustained cytokine up-regulation, corresponding to a chronic remodeling phase.
TABLE 1Up-regulated chemokines and their role aftermyocardial ischemia and reperfusion.Action After Myocardial IschemiaChemokineand ReperfusionCXCL8/Interleukin (IL)-8Induce neutrophil infiltrationCCL2/Monocyte ChemoattractantRegulate monocyte and lymphocyteProtein (MCP)-1recruitmentCCL3/Macrophage InflammatoryRegulate monocyte and lymphocyteProtein (MIP)-1αrecruitmentCCL4/Macrophage InflammatoryRegulate monocyte and lymphocyteProtein (MIP)-1βrecruitmentCXCL10/Interferon-10Angiostatic factor with anti-fibroticproperties[taken from Nah D-Y, Rhee M-Y, Korean Circ. J. October 2009; 39(10): 393-398]
Cell-Mediated Inflammatory Response to Myocardial Infarction
Neutrophils
Neutrophils are recruited during the initial stage of cardiac ischemic injury. Neutrophil transmigration in the infarcted myocardium requires adhesive interactions with activated vascular endothelial cells. Neutrophils may secrete oxidants and proteases and possibly express mediators capable of amplifying cell recruitment [Frangogiannis N G, Youker K A, Entman M L. The role of the neutrophil in myocardial ischemia and reperfusion. EXS. 1996; 76:263-284]. Neutrophil depletion in animals undergoing re-perfused myocardial infarction has been reported to significantly decrease the infarct size, suggesting that a significant amount of myocardial injury may be induced by neutrophil dependent mechanisms [Romson J L, Hook B G, Kunkel S L, Abrams G D, Schork M A, Lucchesi B R. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation. 1983; 67:1016-1023; Jordan J E, Zhao Z Q, Vinten-Johansen J. The role of neutrophils in myocardial ischemia-reperfusion injury. Cardiovasc Res. 1999; 43:860-878].
The mechanisms associated with neutrophil-induced myocardial ischemic injury have not been identified. Jaeschke et al. [Jaeschke H, Smith C W. Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol. 1997; 61:647-653] suggested that neutrophils may directly injure parenchymal cells through release of specific toxic products. While selectins have been implicated, there have been inconsistent results of selectin-related interventions in experimental models of myocardial ischemia [Jones S P, Girod W G, Granger D N, Palazzo A J, Lefer D J. Reperfusion injury is not affected by blockade of P-selectin in the diabetic mouse heart. Am J Physiol. 1999; 277:H763-H769; Birnbaum Y, Patterson M, Kloner R A. The effect of CY1503, a sialyl Lewis X analog blocker of the selectin adhesion molecules, on infarct size and “no reflow” in the rabbit model of acute myocardial infarction/reperfusion. J Mol Cell Cardiol. 1997; 29:2013-2025]. The selectin family consists of L-selectin, P-selectin and E-selectin. P-selectin expression occurs rapidly in endothelial cells during cardiac ischemic injury. Experimental studies have suggested that monoclonal antibodies against P-selectin reduced myocardial necrosis, preserving coronary endothelial function and attenuating neutrophil infiltration in ischemic and reperfused myocardium [Palazzo A J, Jones S P, Anderson D C, Granger D N, Lefer D J. Coronary endothelial P-selectin in pathogenesis of myocardial ischemia-reperfusion injury. Am J Physiol. 1998; 275:H1865-H1872].
Mononuclear Cells
MCP-1/CCL2 μlays an important role in monocyte recruitment to the infarcted myocardium [Dewald O, Zymek P, Winkelmann K, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res. 2005; 96:881-889]. Cytokines, such as TGF-β, free radical oxygen, complement, and the CC chemokines (e.g., MCP-1) may also play a role in monocyte infiltration. Infiltration of monocytes into the infarcted myocardium is followed by maturation and differentiation of these blood-derived cells into macrophages.
Cardiac Repair after Myocardial Infarction
TGF-β as a Key Regulator in Cardiac Repair
TGF-β is a multifunctional cytokine that controls proliferation and cellular differentiation in most cells. The exact role of TGF-β signaling in the infarcted and remodeled heart is poorly understood. Its role in myocardial infarction is thought to involve cardiomyocyte hypertrophy, angiogenic or angiostatic effects, reduced adhesion molecule expression, macrophage deactivation, chemokine and cytokine repression, myofibroblast differentiation, fibroblast proliferation and extracellular matrix protein synthesis [Nah D-Y, Rhee M-Y, Korean Circ. J. October 2009; 39(10): 393-39847; Frangogiannis N G. The immune system and cardiac repair. Pharmacol Res. 2008; 58:88-111].
TGF-β was shown to be significantly up-regulated and TGF-β mRNA and protein was significantly increased at the infarct border zone in an experimental rat model of myocardial infarction [Thompson N L, Bazoberry F, Speir E H, et al. Transforming growth factor beta-1 in acute myocardial infarction in rats. Growth Factors. 1988; 1:91-99; Dean R G, Balding L C, Candido R, et al. Connective tissue growth factor and cardiac fibrosis after myocardial infarction. J Histochem Cytochem. 2005; 53:1245-1256]. During infarct healing, TGF-β may play a role in the suppression of chemokine and cytokine synthesis and is thought to be a key mediator of the transition from inflammation to fibrosis [Bassols A, Massague J. TGF-13 regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans. J Biol Chem. 1988; 263:3039-3045]. Lefer et al. [Lefer A M, Tsao P, Aoki N, Palladino M A., Jr Mediation of cardio-protection by transforming growth factor-beta. Science. 1990; 249:61-64] reported that TGF-β injections reduced myocardial ischemic injury mediated by pro-inflammatory cytokines such as TNF-α during the inflammatory phase of myocardial healing. Anti-TGF-β treatment before or after coronary artery ligation increased mortality and worsened the left ventricular remodeling in mice with non-re-perfused myocardial infarction [Frantz S, Hu K, Adammek A, et al. Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction. Basic Res Cardiol. 2008; 103:485-492]. The inhibition of TGF-β signaling by injection of a TGF-β II receptor resulted in reduction of left ventricular remodeling by modulation of cardiac fibrosis; early TGF-β inhibition increased mortality and left ventricular dilatation [Ikeuchi M, Tsutsui H, Shiomi T, et al Inhibition of TGF-beta signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction. Cardiovasc Res. 2004; 64:526-535; Okada H, Takemura G, Kosai K, et al., Postinfarction gene therapy against transforming growth factor-beta signal modulates infarct tissue dynamics and attenuates left ventricular remodeling and heart failure. Circulation. 2005; 111:2430-2437]. Youn et al. [Youn T J, Kim H S, Oh B H. Ventricular remodeling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade. Basic Res Cardiol. 1999; 94:246-253] reported that an angiotensin converting enzyme inhibitor and angiotensin receptor blockade resulted in decreased TGF-β mRNA expression after non-transmural infarction in the rat.
Other Cytokines in Cardiac Repair
Three IL-1 molecules (IL-1α, IL-β and IL-1 Ra) that are specific receptor antagonists [Dinarello C A. Biologic basis for interleukin-1 in disease. Blood. 1996; 87:2095-2147] have been implicated in cardiac repair. Bujak et al. [Bujak M, Dobaczewski M, Chatila K, et al. Interleukin-1 receptor type I signaling critically regulates infarct healing and cardiac remodeling. Am J Pathol. 2008; 173:57-67] demonstrated that IL-1 signaling is essential for activation of inflammatory and fibrogenic pathways in the healing infarct and plays an important role in the pathogenesis of remodeling after infarction.
IL-10 exerts potent anti-inflammatory effects and modulates MMP expression [de Waal Malefyt R, Abrams J, Bennett B, Figdor C G, de Vries J E. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an auto-regulatory role of IL-10 produced by monocytes. J Exp Med. 1991; 174:1209-1220; Moore K W, deWaal Malefyt R, Coffman R L, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001; 19:683-765; Lacraz S, Nicod L P, Chicheportiche R, Welgus H G, Dayer J M. IL-10 inhibits metalloproteinase and stimulates TIMP-1 production in human mononuclear phagocytes. J Clin Invest. 1995; 96:2304-2310]. However, Zymek et al. [Zymek P, Nah D Y, Bujak M, et al. Interleukin-10 is not a critical regulator of infarct healing and left ventricular remodeling. Cardiovasc. Res. 2007; 74:313-322] reported that IL-10 signaling plays a noncritical role in the suppression of inflammatory mediators, resolution of the inflammatory response and fibrous tissue deposition following myocardial infarction in the mouse; which may be due to the involvement of multiple overlapping regulatory mechanisms controlling various pro-inflammatory pathways activated in the infarcted myocardium.
Proteins in Cardiac Repair
Cluster of differentiation 44 (CD44) is a cell surface glycoprotein involved in cell-cell interaction and cell adhesion and migration. CD44-hyaluronan interactions play a role in leukocyte extravasation at the inflammatory site and serves as a key factor in the resolution of inflammation through removal of matrix breakdown products and clearance of apoptotic neutrophils [Mikecz K, Brennan F R, Kim J H, Glant T T. Anti-CD44 treatment abrogates tissue edema and leukocyte infiltration in murine arthritis. Nat Med. 1995; 1:558-563; DeGrendele H C, Estess P, Siegelman M H. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science. 1997; 278:672-675; Teder P, Vandivier R W, Jiang D, et al. Resolution of lung inflammation by CD44. Science. 2002; 296:155-158]. Huebener et al. [Huebener P, Abou-Khamis T, Zymek P, et al. CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response. J Immunol. 2008; 180:2625-2633] tested the role of CD44 in infarct healing and demonstrated that CD44 mRNA levels were significantly induced in the infarcted heart; CD44 null mice showed enhanced and prolonged inflammation in the infarcted heart followed by decreased myofibroblast infiltration, reduced collagen deposition and diminished proliferative activity. Huebener et al. concluded that CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response.
Thrombospondin (TSP)-1 is a TGF-β activator as well as an adhesive glycoprotein involved in cell-to-cell and cell-to-matrix interaction with potent angiostatic properties [Lawler J. Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med. 2002; 6:1-12]. TSP-1 showed selective localization in the infarct border zone, and TSP-1 knockout animals had markedly increased macrophage and myofibroblast density in the infarct and in remodeling of non-infarcted myocardial areas, and was more extensive in post-infarction remodeling than in wild-type mice. Frangogiannis et al. [Frangogiannis N G, Ren G, Dewald O, et al. The critical role of endogenous thrombospondin (TSP)-1 in preventing expansion of healing myocardial infarcts. Circulation. 2005; 111:2935-2942] concluded that the selective endogenous expression of TSP-1 at the infarct border zone may serve as a “barrier,” limiting expansion of granulation tissue and protecting the non-infarcted myocardium from fibrotic remodeling.
Smad is an essential protein component of the TGF-β pathway [Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003; 113:685-700]. Hao et al. [Hao J, Ju H, Zhao S, Junail A, Scammell-La Fleur T, Dixon I M. Elevation of expression of Smads 2, 3, and 4, decorin and TGF-beta in the chronic phase of myocardial infarct scar healing. J Mol Cell Cardiol. 1999; 31:667-678] showed that TGF-β mRNA was significantly increased in the infarct scar compared to viable myocardium, and that Cardiac Smad 2, 3 and 4 proteins were significantly increased in the border and scar tissues when compared to viable myocardium, suggesting that TGF-β/Smad signaling may be involved in the remodeling of the infarct scar.
The reparative phase of healing involves activation of proteinases, which are critical for cell migration and extracellular matrix remodeling. Recent studies have demonstrated that deficiency of urokinase-type plasminogen activator (uPA) protected mice undergoing left coronary artery ligation against myocardial rupture [Heymans S., Luttun A., Nuyens D., et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 1999; 10:1135-1142]. However, uPA−/− mice also showed impaired scar formation and infarct neovascularization. Furthermore, plasminogen-deficient mice showed a profound disturbance in healing, suggesting a crucial role for the proteolytic system in regulating cardiac repair [Creemers E., Cleutjens J., Smits J., et al. Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction. Am J Pathol 2000; 6:1865-1873].
Matrix metalloproteinase (MMP) expression is upregulated in the infarcted myocardium and may have a prominent role in extracellular matrix remodeling. Administration of MMP inhibitors and targeted deletion of MMP-9 attenuated left ventricular enlargement in murine myocardial infarction [Cleutjens J. P., Kandala J. C., Guarda E., Guntaka R. V., Weber K. T. Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 1995; 6:1281-1292; Lu L., Gunja-Smith Z., Woessner J. F., et al. Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo. Am J Physiol Heart Circ Physiol 2000; 2:H601-H609; Rohde L. E., Ducharme A., Arroyo L. H., et al. Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 1999; 23:3063-30701; Ducharme A., Frantz S., Aikawa M., et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 2000; 1:55-62].
Cardiac Fibroblasts and Extracellular Matrix Remodeling
Cardiac Fibroblasts
In the healthy heart, 70% of the cells present are fibroblasts [Jugdutt B I. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation. 2003; 108:1395-403; Banerjee I, Fuseler J W, Price R L, Borg T K, Baudino T A. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse. Am J. Physiol Heart Circul Physiol. 2007; 293:H1883-91]. Fibroblasts are widely distributed connective tissue cells that are found in all vertebrate organisms. They are usually defined as cells of mesenchymal origin that produce a variety of extracellular matrix (ECM) components, including multiple collagens, as well as fibronectin [Eghbali-Webb M. Molecular Biology Intelligence Unit Molecular Biology of Collagen Matrix in the Heart. Austin, Tex.: Landes; 1994; Kanekar S, Hirozanne T, Terracio L, Borg T K. Cardiac fibroblasts: form and function. Cardiovasc Pathol. 1998; 7: 127-133]. Morphologically, fibroblasts are flat, spindle-shaped cells with multiple processes emanating from the main cell body. Fibroblasts lack a basement membrane, a characteristic feature that separates them from the other permanent cell types of the heart, all of which do contain a basement membrane.
Fibroblasts produce extracellular matrix constituents needed to support cell ingrowth. Willems et al. [Willems I. E., Havenith M. G., De Mey J. G., Daemen M. J. The alpha-smooth muscle actin-positive cells in healing human myocardial scars. Am J Pathol 1994; 4:868-875] previously identified and characterized interstitial nonvascular α-smooth muscle actin (α-SMAc) positive cells, which were present in human myocardial scars 4-6 days after an infarction. These cells are phenotypically modulated fibroblasts, termed myofibroblasts, that develop ultra-structural and phenotypic characteristics of smooth muscle cells and are the predominant source of collagen mRNA in healing myocardial infarcts [Gabbiani G. Evolution and clinical implications of the myofibroblast concept. Cardiovasc Res 1998; 3:545-548]. Myofibroblasts are undifferentiated cells that may be capable of assuming a variety of different roles, such as extracellular matrix metabolism, neovessel formation and contractile activity [Cleutjens J. P., Verluyten M. J., Smiths J. F., Daemen M. J. Collagen remodeling after myocardial infarction in the rat heart. Am J Pathol 1995; 2:325-338; Serini G., Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res 1999; 2:273-283; Cleutjens J. P., Blankesteijn W. M., Daemen M. J., Smits J. F. The infarcted myocardium: simply dead tissue, or a lively target for therapeutic interventions. Cardiovasc Res 1999; 2:232-241]. TGF-β appears to play an important role in myofibroblast differentiation during wound healing by regulating α-SMAc expression in these cells [Desmouliere A., Geinoz A., Gabbiani F., Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993; 1:103-111].
Myofibroblasts are essential for scar formation following myocardial infarction (MI). However, their persistence can contribute to fibrosis and adverse myocardial remodeling, particularly if they remain active in otherwise healthy areas of the heart away from the site of injury. This reactive fibrosis is characterized by increased extracellular matrix and increases the likelihood of arrhythmias [van den Borne S W, Diez J, Blankesteijn W M, Verjans J, Hofstra L, Narula J. Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol. 2010; 7:30-7]. Similarly, the direct coupling of cardiomyocytes to myofibroblasts increases the likelihood of arrhythmias, in contrast to non-activated fibroblasts [Rohr S. Myofibroblasts in diseased hearts: new players in cardiac arrhythmias? Heart Rhythm. 2009; 6:848-56; Thompson S A, Copeland C R, Reich D H, Tung L. Mechanical coupling between myofibroblasts and cardiomyocytes slows electric conduction in fibrotic cell monolayers. Circulation. 2011; 123:2083-93; Rosker C, Salvarani N, Schmutz S, Grand T, Rohr S. Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of alpha-smooth muscle actin containing stress fibers. Circulation Research. 2011; 109:1120-31]. This reactive ongoing fibrosis leads to increased myocardial stiffness that contributes to systolic and diastolic dysfunction and heart failure progression [Pfeffer M A, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81:1161-72; Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev. 1999; 79:215-62]. Over time, the density of myofibroblasts generally decreases following MI, however, these cells can persist in significant numbers for years [Clanachan A S, Jaswal J S, Gandhi M, Bottorff D A, Coughlin J, Finegan B A, et al. Effects of inhibition of myocardial extracellular-responsive kinase and P38 mitogen-activated protein kinase on mechanical function of rat hearts after prolonged hypothermic ischemia. Transplantation. 2003; 75:173-80; Yada M, Shimamoto A, Hampton C R, Chong A J, Takayama H, Rothnie C L, et al. FR167653 diminishes infarct size in a murine model of myocardial ischemia reperfusion injury. J Thorac Cardiovasc Surg. 2004; 128:588-94; Capano M, Crompton M. Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases. Biochem J. 2006; 395:57-64; Aleshin A, Sawa Y, Ono M, Funatsu T, Miyagawa S, Matsuda H. Myocardial protective effect of FR167653; a novel cytokine inhibitor in ischemic-reperfused rat heart. Eur J Cardiothorac Surg. 2004; 26:974-80; Gorog D A, Tanno M, Cao X, Bellahcene M, Bassi R, Kabir A M, et al Inhibition of p38 MAPK activity fails to attenuate contractile dysfunction in a mouse model of low-flow ischemia. Cardiovascular research. 2004; 61:123-31].
After the initial myocardial cell death induced by ischemia, the heart quickly begins to promote the migration of fibroblasts. In the healing wound, fibroblasts proliferate and differentiate into myofibroblasts that take on features resembling smooth muscle cells [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 2005; 45:657-87; Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 2005; 45:657-87; Dobaczewski M, Gonzalez-Quesada C, Frangogiannis N G. The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction. J Mol Cell Cardiol. 2010; 48:504-11].
Extracellular Matrix Remodeling
Remodeling is broadly defined as changes in the organization of the myocardium, and is a critical process that allows the heart to adapt to changes in mechanical, chemical and electrical signals [Brower G L, Chancey A L, Thanigaraj S, Matsubara B B, Janicki J S. Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity. Am J Physiol Heart Circ Physiol. 2002; 283: H518-H525; Chancey A L, Brower G L, Janicki J S. Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function. Am J Physiol Heart Circ Physiol. 2002; 282: H2152-H2158; Stewart J A Jr, Wei C C, Brower G L, Rynders P E, Hankes G H, Dillon A R, Lucchesi P A, Janicki J S, Dell'Italia L J. Cardiac mast cell- and chymase-mediated matrix metalloproteinase activity and left ventricular remodeling in mitral regurgitation in the dog. J Mol Cell Cardiol. 2003; 35: 311-319]. Cardiac fibroblasts are key components of this process, because of their ability to secrete and breakdown the ECM. Degradation of collagen requires the presence of matrix metalloproteinases (MMPs) [Raffetto J D, Khalil R A. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem Pharmacol. 2008; 75: 346-359; Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003; 92: 827-839]. In the normal heart, MMP expression and function are tightly regulated; however, in pathological states, MMP expression and activity are increased, leading to excessive ECM degradation, which can have profound effects on cardiac function. Following cardiac injury, fibroblast function can be influenced by chemical signals (e.g., cytokines, matrikines and growth factors) in a paracrine or autocrine manner. These factors can cause changes in fibroblast gene expression, as well as cell migration to the injured region to promote wound healing and scar formation.
Depending on the stage of heart failure, there can be considerable myocyte hypertrophy and cell death. Dilatation can also be observed in later stages; however, present at every stage are changes in the ECM, which are regulated by cardiac fibroblasts. There is also activation and differentiation of cardiac fibroblasts into myofibroblasts [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 2005; 45: 657-687; Weber K T. Fibrosis in hypertensive heart disease: focus on cardiac fibroblasts. J Hypertens. 2004; 22: 47-50; Frangogiannis N G, Michael L H, Entman M L. Myofibroblasts in re-perfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain. Cardiovasc Res. 2000; 48: 89-100]. After maturation to myofibroblasts, an increase in the synthesis and secretion of fibronectin is observed [Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 2003; 200: 500-503]. As the heart undergoes remodeling associated with heart failure, an increase in cytokine and growth factor secretion is observed. In response to these various factors, myofibroblasts begin to proliferate, migrate and remodel the cardiac interstitium through increased secretion of MMPs and collagen [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure Annu Rev Pharmacol Toxicol. 2005; 45: 657-687; Weber K T. Fibrosis in hypertensive heart disease: focus on cardiac fibroblasts. J Hypertens. 2004; 22: 47-50; Lindsey M L, Escobar G P, Mukherjee R, Goshorn D K, Sheats N J, Bruce J A, Mains I M, Hendrick J K, Hewett K W, Gourdie R G, Matrisian L M, Spinale F G. Matrix metalloproteinase-7 affects connexin-43 levels, electrical conduction, and survival after myocardial infarction. Circulation. 2006; 113: 2919-2928; Raizman J E, Komijenovic J, Chang R, Deng C, Bedosky K M, Rattan S G, Cunnington R H, Freed D H, Dixon I M. The participation of the Na+-Ca2+ exchanger in primary cardiac myofibroblast migration, contraction and proliferation. J Cell Physiol. 2007; 213: 540-551]. To further stimulate the remodeling process, cardiac fibroblasts secrete increased amounts of growth factors and cytokines, specifically IL-1β, IL-6, and tumor necrosis factor (TNF)-α, which, in turn, activate MMPs, leading to further cardiac remodeling [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure Annu Rev Pharmacol Toxicol. 2005; 45: 657-687; Corda S, Samuel J L, Rappaport L. Extracellular matrix and growth factors during heart growth. Heart Fail Rev. 2000; 5: 119-130; Brown R D, Mitchell M D, Long C S. Proinflammatory cytokines and cardiac extracellular matrix: regulation of fibroblast phenotype. In: Villarreal F J, ed. Interstitial Fibrosis in Heart Disease. New York: Springer; 2004: 57-81]. Initially, all of these changes are critical to the reparative wound healing response. However, over time, these changes become maladaptive leading to fibrosis and reduced cardiac function.
Although not present in normal myocardium, myofibroblasts are highly localized to sites of injury where synthesis and deposition of collagen promotes scar formation and fibrosis [Sun Y, Weber K T. Infarct scar: a dynamic tissue. Cardiovasc Res. 2000; 46: 250-256]. In addition, these cells are also located near, or associated with, blood vessels. Because myofibroblasts express contractile proteins, such as smooth muscle actin, they are able to provide mechanical tension to the remodeling matrix, helping to close the wound and reduce scarring [Gabbiani G. The cellular derivation and the life span of the myofibroblast. Pathol Res Pract. 1996; 192: 708-711; Brown E, Dejana E. Cell-to-cell contract and the extracellular matrix. Curr Opin Cell Biol. 2003; 15: 505-508; Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 2003; 200: 500-503]. As the scar matures, cells in the scar undergo apoptosis, leaving a scar that consists mainly of collagen and ECM proteins, but myofibroblasts are still present [Gurtner G C, Werner S, Barrandon Y, Longaker M T. Wound repair and regeneration. Nature. 2008; 453: 314-321]. Myofibroblasts have been observed in mature scars in a rat model of myocardial infarct, as well as in scarred human tissue [Sun Y, Weber K T. Infarct scar: a dynamic tissue. Cardiovasc Res. 2000; 46: 250-256; Willems I E, Havenith M G, DeMey J G, Daemen M J. The alpha-smooth muscle actin-positive cells in healing human myocardial scars. Am J Pathol. 1994; 145: 868-875]. It is not known why myofibroblasts persist, but they are highly involved in regulating cardiac remodeling, cardiac dysfunction, and ultimately cardiac failure.
In the normal heart, collagen and other ECM components help maintain heart structure and function. ECM is synthesized and degraded by cardiac fibroblasts in a coordinated fashion; however, during heart failure there is disruption of these regulatory pathways, leading to an imbalance of ECM synthesis and degradation that determines the level of cardiac remodeling. Increases in the extracellular matrix or fibrosis may be reparative, replacing areas of myocyte loss with a structural scar, or reactive, involving increases in ECM deposition at sites other than those of the primary injury. Fibroblast proliferation and differentiation to myofibroblasts in remote areas of the infarct (reactive fibrosis) can cause an increase in ECM synthesis and deposition which results in increased mechanical stiffness and contributes to relaxation abnormalities, arrhythmogenicity, progressive diastolic dysfunction and heart failure. The size of the infarcted area, the infarcted wound healing, and chronic left ventricular (LV) remodeling determine the extent of heart failure that results [Pfeffer M A, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81:1161-72; Opie L H, Commerford P J, Gersh B J, Pfeffer M A. Controversies in ventricular remodelling. Lancet. 2006; 367:356-67; Dorn G W, 2nd. Novel pharmacotherapies to abrogate postinfarction ventricular remodeling. Nat Rev Cardiol. 2009; 6:283-91]. Progressive increases in fibrosis can lead to systolic dysfunction and left ventricular hypertrophy. Moreover, increased levels of collagen can disrupt electrophysiological communication between myocytes. Furthermore, perivascular fibrosis can impair myocyte oxygen supply, reduce coronary reserve, and accentuate ischemia [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure Annu Rev Pharmacol Toxicol. 2005; 45:657-87].
Anti-fibrosis strategies are limited and are not particularly targeted. Currently, angiotensin-converting enzyme (ACE) inhibition, angiotensin receptor antagonism, and HMG-CoA-reductase inhibition are available [Opie L H, Commerford P J, Gersh B J, Pfeffer M A. Controversies in ventricular remodeling. Lancet. 2006; 367:356-67; Bauersachs J, Galuppo P, Fraccarollo D, Christ M, Ertl G. Improvement of left ventricular remodeling and function by hydroxymethylglutaryl coenzyme a reductase inhibition with cerivastatin in rats with heart failure after myocardial infarction. Circulation. 2001; 104:982-5; Shyu K G, Wang B W, Chen W J, Kuan P, Hung C R. Mechanism of the inhibitory effect of atorvastatin on endoglin expression induced by transforming growth factorbeta1 in cultured cardiac fibroblasts. Eur J Heart Fail. 2010; 12:219-26]. While these have shown some beneficial effects, more effective prevention focused at the level of the fibroblast is needed [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure Annu Rev Pharmacol Toxicol. 2005; 45:657-87; Fraccarollo D, Galuppo P, Bauersachs J. Novel therapeutic approaches to post-infarction remodelling. Cardiovascular research. 2012; 94:293-303].
The Role of the TGFβ/p38 MAPK-MK2 Signaling Pathway in Fibrosis and Post-MI Remodeling
The TGFβ/p38 pathway is central to the pathogenesis of fibrosis. MAPKAP kinase 2 (MK2) is a downstream signaling molecule in the TGFβ/p38 pathway and MK2 phosphorylates and activates signaling molecules that are important in the pathologic processes of fibrotic disease, including inflammatory signaling and fibroblast activation and migration (FIG. 13). MK2 is upstream of both fibrosis and inflammatory pathways. The fibrosis pathway leads to increases in stress fibers (α-smooth muscle actin expression) which results in the myofibroblast phenotype. Myofibroblasts accumulate at sites of tissue remodeling and produce extracellular matrix components such as collagen and hyaluronan (HA) that ultimately compromise organ function. MK2 also phosphorylates transcription factors such as hnRNPA0 which stabilizes cytokines in the inflammatory pathway.
p38 mitogen-activated protein kinase (MAPK) and its upstream and downstream signaling molecules have been shown to play an important role in the response to cellular stress from stimuli [Saklatvala, Curr Opin Pharmacol, 4:372-377, 2004; Edmunds, J. and Talanian, MAPKAP Kinase 2 (MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J and Laufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the Royal Society of Chemistry, 2012].
There are four isoforms of p38 (i.e., p38α, p38β, p38γ, and p38δ) with p38α being most clearly associated with inflammation. Cytokines and other extracellular stimuli (such as growth factors, DNA damage, and oxidative stress) signal through multiple receptors and other mechanisms to activate a cascade of kinases starting with a MAP3K (e.g., MEKK3 or TAK1), then a MAP2K (e.g., MKK3 or MKK6), and then a MAPK (such as p38α). By direct and indirect effects, including the stabilization, translocation, and translation of mRNAs, p38 μlays a major role in the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IFN-γ, as well as the induction of other pro-inflammatory cytokines, such as COX-2.
Generally, in resting cells, p38 MAPK and MK2 are physically bound together in the nucleus. Cellular stress causes the phosphorylation of p38 MAPK by an upstream kinase, such as MKK3 [Kim et al., Am J Physiol Renal Physiol, 292:F1471-1478, 2007]. The activated p38 MAPK then phosphorylates MK2 at residues Thr-222, Ser-272, and/or Thr-334 [Engel et al., EMBO J, 17: 3363-3371, 1998]. The activated MK2 and p38, still physically bound together, translocate to cytoplasm, where they phosphorylate their respective target protein [Ben-Levy et al., Curr Biol, 8:1049-1057, 1998].
In turn, activated MK2 mediates phosphorylation of HSPB1 in response to stress, leading to dissociation of HSPB1 from large small heat-shock protein (sHsps) oligomers, thereby impairing their chaperone activities and ability to protect against oxidative stress effectively. MK2 is also involved in inflammatory and immune responses by regulating Tumor Necrosis Factor (TNF) and IL-6 production post-transcriptionally. This activity is mediated by phosphorylation of Adenine- and Uridine (AU)-Rich Elements (AREs)-binding proteins, such as Embryonic Lethal, Abnormal Vision, Drosophila-Like 1 (ELAVL1), Heterogeneous Nuclear Ribonucleoprotein A0 (HNRNPA0), Polyadenylate-Binding Protein 1 (PABPC1), and Tristetraprolin (TTP/ZFP36), which, in turn, regulate the stability and translation of TNF-α and IL-6 mRNAs. Phosphorylation of TTP/ZFP36, a major post-transcriptional regulator of TNF-α, promotes its binding to 14-3-3 proteins and reduces its affinity to ARE mRNA, thereby inhibits degradation of ARE-containing transcript.
In addition, MK2 also plays an important role in the late G2/M checkpoint following DNA damage through a process of post-transcriptional mRNA stabilization. Following DNA damage, MK2 re-localizes from nucleus to cytoplasm and phosphorylates Heterogeneous Nuclear Ribonucleoprotein A0 (HNRNPA0) and Poly(A)-specific Ribonuclease (PARN), leading to stabilization of growth arrest and DNA-damage-inducible protein 45A (GADD45A) mRNA. Additionally, studies have shown that MK2 is involved in the toll-like receptor signaling pathway (TLR) in dendritic cells and in acute TLR-induced macropinocytosis by phosphorylating and activating Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (RPS6KA3).
Although enzymes at each level of the aforementioned p38 MAPK signaling cascade have been explored for anti-cytokine drug discovery, it is difficult to generalize how upstream or downstream targets in such a pathway might vary in their potential for efficacy. For example, upstream targets might have multiple effects, enhancing efficacy, but might be bypassed by other signaling mechanisms, limiting the impact of inhibition. Undesirable side-effects are similarly difficult to predict. Therefore, specific properties of signaling mechanisms like that of the p38 pathway must be considered case by case to select the best targets based on empirical experience [Edmunds, J. and Talanian, MAPKAP Kinase 2 (MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J and Laufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the Royal Society of Chemistry, 2012].
Indeed, while there have been many reports of p38 inhibitors with promising properties in vitro and in animal models of disease, none have achieved clinical success [Edmunds, J. and Talanian, MAPKAP Kinase 2 (MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J and Laufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the Royal Society of Chemistry, 2012]. Many targets beyond those related to cytokine production are regulated by p38, consistent with observed pleiotropic consequences of its inhibition and suggesting multiple mechanisms of toxicity and even pro-inflammatory effects. For example, in hepatocytes, p38 directly and indirectly down-regulates JNK, thereby modulating hepatocyte sensitivity to lipopolysaccharide (LPS) and TNF-α induced cell death; this may be an important mechanism of p38 inhibition-induced liver toxicity. In addition, activation of MSK1 and MSK2 by p38 may induce expression of anti-inflammatory cytokine IL-10, and therefore inhibition of p38 may have a pro-inflammatory effect that contributes to the observed transient suppression of inflammatory markers by p38 inhibitors. Thus, there are significant concerns that, as an anti-inflammatory strategy, p38 inhibition will not result in adequate efficacy or acceptable safety.
On the other hand, MK2 attracted wide attention as a potential drug discovery target when it was reported that MK2-deficient knockout mice are viable and fertile, and are defective in TNF-α production. Splenocytes derived from these animals are defective in the production of several pro-inflammatory cytokines, including TNF-α, IL-6 and IFN-γ and the animals themselves are resistant to collagen-induced arthritis (a mouse model of rheumatoid arthritis (RA)), as well as in ovalbumin-induced airway inflammation (a mouse model of asthma). Dosed orally, inhibitors of MK2 can block acute systemic induction of TNF-α by LPS in rats and can reduce paw swelling in the rat streptococcal cell wall (SCW)-induced arthritis model. These results suggested that MK2 mediates most or all inflammatory signals of the p38 cascade while other p38 substrates regulate the pathways responsible for toxicity or attenuated efficacy; and that MK2 inhibition might deliver on the promise of p38 inhibition for anti-inflammatory efficacy while also giving a more favorable safety profile.
Myocyte death during lethal myocardial infarction, cardiac dysfunction, and fibrosis during post-MI remodeling and hypertrophy is associated with sustained activation of p38 [Clark J E, Sarafraz N, Marber M S. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther. 2007; 116:192-206; Kerkela R, Force T. p38 mitogen-activated protein kinase: a future target for heart failure therapy? Journal of the American College of Cardiology. 2006; 48:556-8; Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation. 2007; 116:1413-23]. Recent studies in MK2−/− mice have illustrated that MK2 acts downstream of p38 and is responsible for p38-induced heart failure [Streicher J M. The role of mitogen activated protein kinase activated protein kinase-2 in regulating p38 mitogen activated protein kinase induced cyclooxygenase-2 induction and heart failure: University of California-Los Angeles; 2009]. Similarly, MK2−/− mice are resistant to ischemia reperfusion injury [Shiroto K, Otani H, Yamamoto F, Huang C K, Maulik N, Das D K. MK2−/− gene knockout mouse hearts carry anti-apoptotic signal and are resistant to ischemia reperfusion injury. J Mol Cell Cardiol. 2005; 38:93-7], indicating a critical role of MK2 in ischemic heart disease experimentally. When mice lacking MK2 (MK2−/−) were compared to M2+/+ mice on a transgenic p38 background, the transgenic p38-induced heart failure in the MK2−/− mice was significantly protective [Streicher J M. The role of mitogen activated protein kinase activated protein kinase-2 in regulating p38 mitogen activated protein kinase induced cyclooxygenase-2 induction and heart failure: University of California-Los Angeles; 2009]. Similarly, MK2−/− mice are resistant to ischemia reperfusion injury [Shiroto K, Otani H, Yamamoto F, Huang C K, Maulik N, Das D K. MK2−/− gene knockout mouse hearts carry anti-apoptotic signal and are resistant to ischemia reperfusion injury. J Mol Cell Cardiol. 2005; 38:93-7], implicating a critical role of MK2 in ischemic injury. Consistent with an MK2-p38 axis mediating ischemic cardiac damage, inhibiting p38 activation protects the heart against ischemic insult and cardiac dysfunction [Marber M S, Rose B, Wang Y. The p38 mitogen-activated protein kinase pathway—a potential target for intervention in infarction, hypertrophy, and heart failure. J Mol Cell Cardiol. 2011; 51:485-90; Tanno M, Bassi R, Gorog D A, Saurin A T, Jiang J, Heads R J, et al. Diverse mechanisms of myocardial p38 mitogen-activated protein kinase activation: evidence for MKK-independent activation by a TAB1-associated mechanism contributing to injury during myocardial ischemia. Circulation Res. 2003; 93:254-61; Marais E, Genade S, Huisamen B, Strijdom J G, Moolman J A, Lochner A. Activation of p38 MAPK induced by a multi-cycle ischaemic preconditioning protocol is associated with attenuated p38 MAPK activity during sustained ischemia and reperfusion. J Mol Cell Cardiol. 2001; 33:769-78; Sanada S, Kitakaze M, Papst P J, Hatanaka K, Asanuma H, Aki T, et al. Role of phasic dynamism of p38 mitogen-activated protein kinase activation in ischemic preconditioning of the canine heart. Circulation Res. 2001; 88:175-80; Nagarkatti D S, Sha'afi R I. Role of p38 MAP kinase in myocardial stress. J Mol Cell Cardiol. 1998; 30:1651-64]. At the cellular level, ischemic activation of the MK2-p38 signalling pathway induces cardiac apoptosis [Matsumoto-Ida M, Takimoto Y, Aoyama T, Akao M, Takeda T, Kita T. Activation of TGF-beta1-TAK1-p38 MAPK pathway in spared cardiomyocytes is involved in left ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circul Physiol. 2006; 290:H709-15], specifically in cardiomyocytes [Clark J E, Sarafraz N, Marber M S. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther. 2007; 116:192-206; Kerkela R, Force T. p38 mitogen-activated protein kinase: a future target for heart failure therapy? Journal of the American College of Cardiology. 2006; 48:556-8; Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation. 2007; 116:1413-23]. In fibroblasts, p38 regulates extracellular matrix proteins in primary cardiac fibroblasts during oxidative stress [Hsu P L, Su B C, Kuok Q Y, Mo F E. Extracellular matrix protein CCN1 regulates cardiomyocyte apoptosis in mice with stress-induced cardiac injury. Cardiovascular Res. 2013; 98:64-72].
The use of rationally designed cell-permeable peptides that inhibit Mitogen Activated Protein Kinase Activated Protein Kinase II (MK2) activity and downstream fibrosis and inflammation is unique. Recent studies have reported that the cell-permeable peptide MMI-0100 inhibits inflammation and fibrosis in intimal hyperplasia in a mouse vein graft model [Muto A, Panitch A, Kim N, Park K, Komalavilas P, Brophy C M, et al Inhibition of Mitogen Activated Protein Kinase Activated Protein Kinase II with MMI-0100 reduces intimal hyperplasia ex vivo and in vivo. Vascular Pharmacol. 2012; 56:47-55], bleomycin-induced pulmonary fibrosis [Vittal R, Fisher A, Gu H, Mickler E A, Panitch A, Lander C, et al. Peptide-mediated Inhibition of MK2 Ameliorates Bleomycin-Induced Pulmonary Fibrosis. Am J Respir Cell Mol Biol. 2013] and in inhibiting abdominal adhesions post-surgery [Ward B C, Kavalukas S, Brugnano J, Barbul A, Panitch A. Peptide inhibitors of MK2 show promise for inhibition of abdominal adhesions. J Surg Res 2011; 169:e27-36]. These peptides target the substrate-binding site of MK2 and contain permeant domains that are rapidly taken up by macropinocytosis and targeted to endosomal compartments, where they are retained for up to 7 days [Flynn C R, Cheung-Flynn J, Smoke C C, Lowry D, Roberson R, Sheller M R, et al. Internalization and intracellular trafficking of a PTD-conjugated anti-fibrotic peptide, AZX100, in human dermal keloid fibroblasts. J Pharm Sci. 2010; 99:3100-21].
To minimize the extent of heart failure after a large or recurrent MI, therapeutic strategies are needed to limit infarct wound healing. The described invention offers approaches to minimize the extent of heart failure or recurrent MI by utilizing a cell-penetrating, peptide-based inhibitor of MK2.